Studies examining effects of ethanol and/or aging on membrane fluidity and composition have reported on changes in the bulk membrane. However, the membrane consists of two monolayers or leaflets that do not appear to be coupled and are structurally and compositionally asymmetric. Data on ethanol-induced changes and changes with age have been an average of the two leaflets. Using a new method to determine membrane domains, we have recently found that the outer leaflet of synaptic plasma membranes (SPM) is more fluid as compared to the inner leaflet and that ethanol in vitro had a greater effect on the outer leaflet. This asymmetric effect of ethanol may be necessary for transmembrane function in response to ethanol, e.g., enzyme activity, transport, receptoreffector complex. Such an effect may not be present in membranes of aged animals as a result of age changes in membrane asymmetry resulting from changes in lipid distribution between the two leaflets with increasing age. This hypothesis will be tested using biophysical and biochemical procedures to examine the transbilayer structural and compositional properties of SPM from 6, 18, and 30 month old C578BL/6NNIA mice. The experiments in this application will address the following issues: 1) What are the effects of ethanol in vitro (i.e., fluidity) and in vivo (i.e., fluidity, lipid distribution) on the outer and inner leaflets of SPM?; 2) Does fluidity and lipid asymmetry change with increasing age and are there age differences in effects of ethanol in vitro and in vivo on the individual membrane leaflets?; 3) What is the duration of ethanol- induced changes in SPM leaflets upon withdrawal and is this age- related?; and 4) Will modification of membrane asymmetry alter age differences in response to ethanol. The proposed work will examine for the first time the effects of ethanol on the outer and inner membrane leaflets. It is our intention that the results of the proposed studies will provide new insight into effects of ethanol on membrane domains, data on age changes in membranes and procedures that might alter changes in membranes induced by ethanol and aging.